Abstract: The overarching objective of the proposed studies is to identify and characterize early premalignant changes in breast tissues from women that carry genetic alterations associated with a high risk of breast cancer to ultimately develop strategies to detect and prevent the development of breast cancer. To accomplish this goal, we have optimized three technologies to profile single breast mammary cells (MECs): (1) CyTOF mass cytometry to allow tracking in parallel of >30 cell lineage and proliferation markers, (2) single cell RNA sequencing to identify expression programs of cell populations enriched in mutation-carriers, and (3) multi-plex cyclic immunofluorescence imaging (CyCIF) to simultaneous image >50 markers in situ. These technologies make it possible to detect differences in small populations of cells that would be masked by bulk population analyses. To date, we have profiled breast tissues from over 30 women with wild-type or mutant BRCA1 or BRCA2 by CyTOF and have identified distinct, previously unrecognized subpopulations of cells that are enriched in breast tissues from BRCA1 and/or BRCA2 carriers. These enriched subpopulations may represent cells that are either directly on the path to malignancy or indirectly contribute to the development of cancer in these high-risk women. We have identified RNA signatures associated with these enriched subpopulations, which include surface markers to isolate them from breast tissue to investigate both these possibilities and to track them within breast tumors. The signatures associated with one of the enriched populations have provided clues as to the basis for their accumulation, as well as potential strategies to prevent their accumulation. Using CyCIF, we have been able to identify enriched subpopulations of cells in situ within breast tissues and track their association with aberrant histologies. We have also developed organoid cultures that maintain all of the major MEC lineages as well as the BRCA1/2-enriched populations, and that are able to reconstitute glandular structures in immunocompromised mice. We believe that these tools provide an unprecedented opportunity to track the development of human cancer. In the proposed studies, we will investigate whether and how the BRCA1/2+/mut-enriched subpopulations contribute to tumorigenesis in mutation carriers. We will also investigate the basis for the enrichment of these populations and the contribution of DNA damage to their enrichment. Later stage studies will focus on the development of strategies to interfere with tumor progression, and importantly to develop novel diagnostic strategies to inform on the timing of prophylactic interventions. In addition, we will examine tissues from women who carry mutations in other breast cancer predisposition genes to establish whether similar subpopulations are detected in other high-risk individuals. And lastly, we will examine the possibility that these cells represent cells-of-origin of sporadic breast tumors that arise more broadly in the population.